1. Field of the Invention (Technical Field)
The present invention relates to a particle analyzer and sample handling for use with particle analysis apparatus and a method for high-throughput analysis using the same.
Particle analysis using flow cytometry is used to characterize cells and particles by making measurements on each at rates up to thousands of events per second. The measurement consists of simultaneous detection of the light scatter and fluorescence associated with each event. Commonly, the fluorescence characterizes the expression of cell surface molecules or intracellular markers sensitive to cellular responses to drug molecules. The technique often permits homogeneous analysis such that cell associated fluorescence can often be measured in a background of free fluorescent indicator. The technique often permits individual particles to be sorted from one another.
However, a deficiency with conventional flow cytometry is that it does not allow for the analysis of multiple samples consisting of multiple cells or particles in a rapid manner, a fact that has limited the uses of flow cytometry in drug discovery and other high throughput screening applications. For example, the industrial standard for high throughput drug discovery is 100,000 samples per day. Because of its low throughput, flow cytometry has generally not been considered applicable to high throughput screening applications in areas such as drug discovery, antibody hybridoma screening, and systems biology.
There have been several efforts at automated sample handling in flow cytometry. For example, sample handling systems are known that use carousels to handle samples from standard sized tubes and sample injection systems which handle samples from 96-384 well microplates. These systems treat each tube or well as a single sample. A separate data file is created for each sample. These systems typically intake samples at a rate of approximately 1 up to 5 samples per minute and require priming the sample line with each individual specimen before analysis. Therefore, a single data file exists for each well sample interrogated.
Other groups have also used valves and syringes in flow cytometry, most notably, the “flow injection” group, Lindberg et al. at University of Washington. However, the processes described above did not address throughput speed. A group at the University of New Mexico has used high throughput flow cytometry and achieved sampling rates as high as 40 samples per minute, see U.S. Pat. Nos. 6,878,556, 6,890,487, 7,368,084, the entire disclosure and contents of which is hereby incorporated by reference. However, to our knowledge, no one has reported the capability to process samples at rates higher than 40 samples per minute for flow cytometry.
The presence of uncontrolled bubbles in the flow cytometer system is one of the primary sources of corrupted experimental data. Flow cytometers may periodically experience bubbles in the sheath fluid or sample fluid lines. Precautions are taken to exclude bubbles as bubbles are known to cause anomalies in the flow within the flow cytometer system that reduce the performance of the flow cytometer. Furthermore, bubbles passing through the interrogation zone of the flow cytometer can cause spurious or false event signals that corrupt the experimental data being collected. The user can take corrective action only after the bubbles have been detected, which often occurs after experimental data has been corrupted and the user has been inconvenienced. Therefore the flow cytometry art teaches against introduction of bubbles in the flow stream.